Abstract Mediator, a large (25 polypeptides, MW ~1 MDa) multi-protein complex conserved throughout eukaryotes is the key coactivator responsible for conveying regulatory signals to RNA polymerase II (RNAPII) during transcription initiation. Mediator has essentially no enzymatic activity and it is generally agreed that its mechanism must be based on modulation of molecular interactions and conformational rearrangements. Therefore, a structural understanding of Mediator and its interaction with RNAPII is essential to elucidate how Mediator's role during initiation ultimately influences cell differentiation, development, and homeostasis. Macromolecular electron microscopy (EM) is the technique of choice for characterization of large, dynamic macromolecular assemblies because it is uniquely suited to provide information about their structure, conformational changes, and interactions. EM studies of Mediator supported by this grant have set the stage to achieve a molecular understanding of the complex and here we describe further EM, biochemical, and functional analyses of yeast and human Mediators that build on our previous work and will provide a detailed understanding of the structure, subunit organization, and regulation of Mediator conformation and interactions, and reveal the mechanism of regulation by Mediator across eukaryotes. In Aim 1 we will pursue EM analysis of yeast Mediator (yMED) at subnanometer resolution and investigate subunit interfaces critical for yMED assembly, conformational variability, and function. In Aim 2 we will study how different factors control Mediator conformation and interactions to modulate RNAPII engagement and transcription initiation, and obtain detailed structural information about the Mediator-RNAPII holoenzyme. In Aim 3 we will leverage the experience gained from EM analysis of yMED to pursue EM studies of the structure, subunit organization, and interactions of human Mediator (hMED). Parallel analysis of yeast and human Mediators will reveal fundamental aspects of the regulation mechanism related to structural conservation of Mediator across eukaryotes, while highlight aspects of regulation specific to the more intricate human complex. Our results will provide a molecular understanding of the way in which Mediator enables regulation of transcription initiation, and help us understand the direct connection between dysregulation of gene expression and oncogenesis. The work we propose continues our strategy of combining structural analysis with biochemical and functional studies, and will depend critically on 1) application of novel EM image analysis approaches developed in collaboration with the group of Pawel Penczek (UT Houston) specifically tailored to study the structure of dynamic complexes; 2) a strong, ongoing collaboration with the group led by Joan and Ron Conaway (Stowers Institute), leaders in biochemical and functional studies of human Mediator.